GOLGI LOCALIZATION AND FUNCTIONAL EXPRESSION OF HUMAN URIDINE DIPHOSPHATASE

A full-length E(ecto)-ATPase (Plesner, L. (1995) Inf. Rev. Cytol, 158, 141-214) cDNA was cloned from a human brain cDNA library; it encodes a 610-amino acid protein that contains two putative transmembrane doma ins. Heterologous expression of this protein in COS-7 cells caused a s ignificant increase in intracellular membrane-bound nucleoside phospha tase activity. The activity was highest with UDP as substrate and was stimulated by divalent cations in the following order: Ca2+ much great er than Mg2+ > Mn2+. The results of immunofluorescence staining indica te that this protein is located in the Gels apparatus. UDP hydrolysis was increased in the presence of Triton X-100 or alamethicin, an ionop hore that facilitates movement of UDP across the membrane, suggesting that the active site of this UDPase is on the luminal side of the Golg i apparatus. This is the first identification of a mammalian Golgi lum inal UDPase gene. Computer-aided sequence analysis of the E-ATPase sup erfamily indicates that the human UDPase is highly similar to two hypo thetical proteins of the nematode Caenorhabditis elegans and to an uni dentified 71.9-kDa yeast protein and is less related 60 the previously identified yeast Golgi GDPase.